Radiol Oncol 2009; 43(4): 282-292.
doi:10.2478/v10019-009-0034-8
research article
Influence of surgical treatment and radiotherapy of the advanced intraoral cancers on complete blood count, body mass index, liver enzymes and leukocyte CD64 expression Tadej Dovšak1, Alojz Ihan2, Vojislav Didanovič1, Andrej Kansky1, Nataša Ihan Hren1 1Clinical
Department of Maxillofacial and Oral Surgery, University Medical Center, Ljubljana, Slovenia; 2Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Slovenia
Background. The aim of our study was to evaluate the influence of the surgery and radiotherapy of the advanced oral squamous cell carcinoma on the complete blood count, body mass index (BMI), acute inflammatory response, liver enzymes and expression of the CD64 index on leukocytes in the peripheral blood. Patients and method. Venous blood was obtained from 16 patients with advanced oral squamous cell carcinomas treated with radical surgery and external beam radiotherapy. Blood samples were collected prior to surgery (T1), after surgery (T2) and after radiotherapy (T3). Blood samples were analyzed for whole blood count, immunoglobulin G levels, liver enzymes (transaminases (ALT and AST) and gamma-glutamyl trasferase (γ-GT)), inflammatory response markers (C-reactive protein, erythrocyte sedimentation rate, albumin, white blood count, leukocyte count and CD64 expression on leukocytes). Assessment of nutrition was done by calculating the body mass index. Results. Surgery caused anaemia, trombocytosis, leukocytosis, lymphopenia, rise in acute phase proteins, elevation of CD64 expression on monocytes and neutrophyls, elevation of liver transaminases and lowering of γ-GT, albumin, protein and bilirubin levels. After radiotherapy haemoglobin, leukocytes, C-reactive protein, erythrocyte sedimentation rate, liver transferases, albumin, bilirubin and proteins returned almost to T1 levels, levels of lymphocytes, γ-GT and body mass index lowered. IgG levels remained almost unchanged at T2 and T3. Levels of the CD64 expression on monocytes and neutrophyls also elevated after radiotherapy. Conclusions. Surgery caused a significantly larger acute phase response than radiotherapy, while radiotherapy worsened the already present lymphopenia. Key words: intraoral cancer; surgery; radiotherapy; blood parameters Received 16 July 2009 Accepted 11 August 2009 Correspondence to: Tadej Dovšak, MD, Clinical Department of Maxillofacial and Oral Surgery, University Medical Center, Ljubljana, Slovenia; Phone: +386 1 522 4235; Fax: +386 1 522 2495; E mail: tadej.
[email protected]
Dovšak T et al. /Intraoral cancer and blood parameters after the therapy
Introduction Oral squamous cell carcinoma (OSCC) is the sixth most common neoplasm in the European Union.1 Advanced OSCC is treated with surgery and then radiotherapy (RT) and/or chemotherapy.2 Five year survival rates for patients with advanced OSCC are below 55%3, so researchers are still searching for better treatment modalities. The single most important factor influencing the outcome of patients with squamous cell carcinomas (SCC) of the upper aerodigestive tract is like at the other malignancies the stage of the disease at the time of initial diagnosis and treatment.4-6 Once the tumour is no longer localized but has disseminated to regional lymph nodes, the probability of 5 year survival reduces to nearly half.7-9 The patient’s long-time prognosis is also worsened by the fact that the new primary SCC of upper aerodigestive tract occurred in 20% of these patients.10 Surgical trauma produces alterations in the hemodynamic, metabolic and immune responses of patients in the postoperative period. This injury response is a dynamic process that follows a specific pattern that has been defined based on clinical and scientific observations.11 The initial proinflammatory immune response or systemic inflammatory response syndrome (SIRS) is mediated primarily by the cells of innate immune system. This is followed by a compensatory anti-inflammatory or immunosuppressive phenotype that is mediated primarily by cells of the adaptive immune system, which predisposes the host to septic complications9 and may be also promotes the tumour growth and metastases.13,14 Cell mediated immunity is suppressed for several days after surgery and more invasive procedures lead to deeper and longer immunosuppression.14,15 The role of RT for OSCC treatment is well established; modern equipment and
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techniques have minimized morbidity.16 Toxicity is related to site and dose; acute toxicity is related to the inflammatory process induced within the radiation field. These effects occur to some degree in the majority of patients, but they are self-limited in duration. The presence of late toxicity is determined by the total dose of radiation given and not by the daily dose or fraction size.17-19 Although lymphocytes are highly sensitive to radiation damage19,20 this toxicity is not usually assessed. The aim of our study was to evaluate the effect of major surgical procedures and RT of advanced OSCC on the complete blood count, body mass index (BMI), acute inflammatory response, liver enzymes and expression of the CD64 index on leukocytes in the peripheral blood by the prospective non-randomized study.
Patients and methods Our prospective non-randomized study was running from 2007 to 2009 on the Clinical Department for Oral and Maxillofacial Surgery, University Clinical Center in Ljubljana. Sixteen patients with advanced OSCC were selected. The study group included 12 men and 4 women with median age 61 years (range 42-80 years). All were Stage III and IV according to the American Joint Committee on Cancer staging.21 Inclusion criteria were that surgery and radiotherapy were the only treatment modalities. The local Ethics Committee of the Republic of Slovenia ensured that research protocol and appropriate written consent was obtained from each patient. Blood samples T1 were collected between 1 to 17 days before surgery (mean ± SD; 8.9 ± 5.9). All patients were surgically treated with en bloc excision of tumour and modified neck dissection (five of them with bilateral) and subsequent reRadiol Oncol 2009; 43(4): 282-292.
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Dovšak T et al. /Intraoral cancer and blood parameters after the therapy
construction with flaps (9 with free flaps: 6 radial forearm, 1 iliac crest, 1 anterior lateral thigh, 1 fibula; 3 with pedicled flaps: 2 pectoralis major muscle, 1 temporalis muscle). Median blood loss during operation was 430 ml (range 200-1000 ml) which was assessed by the anaesthesiologist and surgeon. In all but one patient, temporary tracheotomy was performed at the time of operation. All patients had confirmed clear margins with frozen sections at the time of operation. In one patient, revision of the operative field was required immediately after operation because of haemorrhage. All free flaps were viable and functioning. The tracheotomy tube was removed at median day 6 (range 3-15). All patients were fed by nasogastric tubes (NGTs) for the median time of 9 days (range 5-15). Six patients received postoperative transfusion of concentrated erythrocytes for the correction of haemoglobin levels bellow 90 mg/l. All patients were treated with antibiotics for the median time of 8 days (range 5-10). Blood samples T2 were taken between 8 and 26 days after surgery (mean ± SD: 15.4 ± 4.4). Patients were irradiated with an external beam on the 6 MV linear accelerator. They received between 58 and 66 Gy (mean ± SD: 60.5 ± 1.9), divided on 2 Gy daily fractions, five times a week. This RT was applied within 6 weeks after the surgery. No patient received hyperfractionated RT or chemotherapy. Blood samples T3 have been collected from 28 days up to 128 days postRT (mean ± SD: 56.6 ± 36.9). At the time of blood sampling all patients were weighted and their height was measured at T1. These measures were used to calculate body mass index (BMI, kg/m2).
Blood sampling The blood samples were taken from the cubital vein. 1 EDTA-containing test tube (5 ml) of peripheral blood was obtained Radiol Oncol 2009; 43(4): 282-292.
for haematological, biochemical and cytometrical laboratory tests.
Expression of CD64 on neutrophils and monocytes Expression of CD64 on neutrophils, monocytes and lymphocytes was measured by quantitative flow cytometry with a FACSCalibur flow cytometer (Becton Dickinson, NY, USA) and FACSCanto flow cytometer (Becton Dickinson, CD, USA) using the Leuko64TM assay (Trillium Diagnostics, LLC, Maine, USA). The assay is for research use only and is composed of three antibodies with specificities to CD64 (clones 22 and 32.2, both fluorescein isothiocyanate (FITC) conjugated) and a fluorescence bead suspension with three fluorescence signals (green fluorescence due to FITC, orange fluorescence similar to PE and red fluorescence of starfire red) for unique identification of beads, and used for instrument calibration and standardization of leukocyte CD64 expression in human blood. The sample preparation and flow cytometer setup were based on the manufacturer’s instructions. Briefly, 50 μL of whole blood, or diluted whole blood to adjust leukocyte concentration to less than 25 x109 /L, was incubated for 15 minutes in the dark at room temperature with a mixture of murine monoclonal antibodies followed by red cell lyses with an ammonium-chloride-based solution (Trillium Lyse). Fluorescence beads were then added and flow cytometer analysis was performed on a minimum of 50,000 leukocytes. Data analysis for fluorescence intensity was performed by CellQuest software (Becton Dickinson, CA, USA). MFI was measured as a linearized value of log scale on monocytes (green, positive control, measuring CD64 expression), neutrophils (blue, measuring CD64 expression), and beads (aqua blue, measuring FITC and PE expres-
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Table 1. Median values; SD of leukocytes, platelets, neutrophils and lymphocytes (10*9/L), erythrocytes (10*12/L) and Hb levels (g/L) in observed times (before surgery (T1) and after surgery (T2); after RT – T3) and their normal values. The significant differences (p
